ABSTRACT Senescent cells, marked by persistent p16INK4a expression, increase in aging tissues and promote the onset of age-related disease in mice. These observations have led to widespread pre-clinical and clinical use of p16INK4a as a surrogate marker of declining biological function. However, the age-related molecular events that cause p16INK4a levels to rise are largely undefined and studies have yet to determine if senescent cells retain any normal physiological functions. Using an innovative technique to simultaneously profile mRNAs indicative of T-cell subtype, function and cellular senescence, we find that p16INK4a levels rise in parallel with markers of T-cell exhaustion. Exhaustion is a progressive form of T-cell dysfunction catalyzed by repeated immune stimulation. Exhausted T-cells express inhibitory receptors that oppose T-cell receptor (TCR) signaling when activated by exogenous ligands. Current and emerging cancer immunotherapies block these receptor-ligand interactions, known as immune checkpoints, to restore the function of tumor-specific T-cells. We observe that the expression of p16INK4a in peripheral blood T-lymphocytes (PBTLs) from newly diagnosed melanoma patients is age-independent and appears to predict immunotherapeutic discontinuation. These findings support our overarching hypothesis that T-cell senescence is a consequence of continued antigenic stimulation and dictates the outcome of immune checkpoint inhibitor therapy in melanoma. We propose a model wherein initial T-cell stimulation leads to the transient upregulation of both p16INK4a and markers of T-cell exhaustion. However, when the TCR is repeatedly engaged, sustained p16INK4a expression drives entry into cellular senescence, thereby locking T-cells into a non- proliferative state in which immunotherapies can no longer provide benefit to the patient. The proposed studies will determine the chronologic, molecular and functional relationship between T-cell exhaustion and cellular senescence (Aim 1A, C), identify the physiological processes that regulate p16INK4a downstream of TCR activation (Aim 1B) and define the impact of T-cell senescence in immune checkpoint inhibitor therapy (Aim 2). This work will delineate the physiological events that trigger T-cell senescence and establish the identity and functional capacity of these cells, which are increasingly used as a surrogate measure of biological aging. These studies are also likely to advance cancer immunotherapy regimens by demonstrating that a circulating peripheral blood marker could be used to predict outcome, improve response rates and minimize toxicity.